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Abstract

Objective

The balance of oral microbiota is important in oral disease prevention and treatment. Oral microbiomes play an essential role in oral diseases through anti-inflammatory effects and restoring dysbiosis. Lactococcus lactis subspecies lactis strains Y-PDH01-07 (LL01-07) were identified from dadih, a traditional Indonesian fermented milk. Previous research had shown that dadih can reduce colon inflammation. However, the characteristics of LL01-07 as an oral probiotic have not been explored. The objective of this research is to characterize and functionally assess the potential of LL01-07 as an oral probiotic.

Methods

Strains were identified using phenotypic features, PCR, and sequencing, and their biosafety status was evaluated. The oral probiotic characterization of LL01-07 consisted of carbohydrate fermentation ability, antimicrobial activity, auto-aggregation, hydrophobicity, and acid resistance. An in vivo experiment was conducted to evaluate its anti-inflammatory effect on the oral mucosal wound model with unpaired t-test to evaluate the group's significance.

Results

Genotypic results confirmed that all isolates were Lactococcus lactis subsp. lactis, and biosafety assessment showed no inhibition zone in antibiotic resistance test and no hemolytic activity. LL05 had the highest % auto aggregation: 15.0 ± 1.7% at 33 h and 54.2 ± 4.8% at 22 h. LL05 had the best acid resistance test (75.2% survival after 4 h of acid exposure). The lesion severity score did not improve in the treatment group, but body weight increased.

Conclusions

LL 01-07 was safe to administer to oral cavity but showed less oral probiotic potential. The in vivo study showed that the treatment group had a lower wound healing effect score but a higher average body weight.

Keywords

dentistry oral probiotic buccal wound healing

Introduction

The oral cavity is important for speech and mastication; hence, the presence of oral diseases can cause significant pain and financial burden. The oral cavity is inhabited by more than 700–1000 species of bacteria.¹ The increase of pathogenic bacteria and the decrease of beneficial microbes trigger oral disease.² Oral diseases, such as caries, periodontal disease, stomatitis, and mucositis, are prevalent and often neglected despite their considerable pain and high cost, lowering the patients’ quality of life and productivity.³ These oral problems often cause poor mastication, leading to malnutrition. Furthermore, oral inflammation predisposes patients to chronic low-grade systemic inflammation.⁴

Oral mucositis is an oral disease related to the side effects of cancer therapy and is characterized by painful mucosal atrophy, erosion, ulceration, or a combination of these.¹ The loss of mucosal integrity becomes a port of microbial entry, resulting in opportunistic infection or even septicemia in neutropenic patients.^1,5 Moreover, they might have delayed cancer treatment and a worse prognosis.⁵ Dysbiosis was recognized following anticancer administration and along with mucositis manifestation.⁶ It alters homeostasis of the oral environment and aggravates oral mucositis severity.⁷

The oral microbiota balance plays an important role in oral disease prevention and treatment. Microbial-based regimens, such as chlorhexidine, antibiotics, and probiotics, have been used to reduce the microbiota burden. Chlorhexidine has side effects, including altered taste.⁷ Use of antibiotics disrupts the commensal microbiota, resulting in higher toxicity.^7,8 Oral probiotics have become a promising strategy for improving oral health.¹ It improves the balance between symbionts and pathobionts, thus initiating cytoprotective pathways, modulating local inflammation, and increasing the mucosal barrier permeability.⁷ Increasing studies on probiotic use for periodontitis,⁹ and oral mucositis showed positive results in decreasing its severity and duration.¹⁰

Probiotics, according to WHO in 2002, are “live microorganisms which, when administered in adequate amounts, confer benefits to the health of the host”.^1,11 The screening of probiotic candidates must consider the safety status of the bacteria and be sufficiently characterized.^12,13 Generally, a guideline for evaluating a potential probiotic candidate starts with the identification of the bacterial species and strain, followed by safety assessment and functional characterization. Note that in vitro data should be validated using in vivo evaluation.¹¹ Some important characteristics of oral probiotics are hampering oral pathogen growth, adhering to and colonizing oral surfaces, non-sugar fermenting, not declining oral pH, inhibiting the oral biofilm, and minimizing the cytotoxic products from pathogenic bacteria. Importantly, oral probiotics must be safe for the host.¹⁴

Lactococcus lactis subsp. lactis strain Y-PDH01-07 (LL01-07) is the most abundant strain found in dadih, a traditional milk fermentation product from Agam, West Sumatra. Dadih could harbor many bacteria and had been known for a long time for many health benefits. Study shows it has anticholesterol, antimicrobial, and antioxidant activities.¹⁵ Dadih demonstrated the ability to modulate inflammatory mediators and suppress the inflammation severity score in the mouse colon inflammation model.¹⁶ In oral health, topical application of dadih was able to reduce gingival inflammation.¹⁷ Lactococcus lactis is “generally recognized as safe” (GRAS) due to its historical use, and some natural isolates have anti-inflammatory properties.¹⁸ L. lactis had been investigated for its benefits in oral health. L. lactis Strain NCC2211 can modulate cariogenic bacteria.³ Combination of L. lactis and Streptococcus thermopillus has antimicrobial activity towards Porphyromonas gingivalis, and could reduce inflammatory cytokine in periodontitis.¹⁹

The exploration of the strains’ ability to support oral wound healing would be a significant contribution to support patient's quality of life. Therefore, the development of local topical probiotic products started with the characterization and evaluation of LL01-07 as a topical probiotic to enhance the healing of oral diseases including oral mucositis, is needed. To our knowledge, no local probiotic product has been developed for oral disease in Indonesia.

The objective of this study was to characterize the dadih-derived isolates, LL01-07 used for oral wound healing. The first step is the phenotypic and genotypic identification of dadih-derived strains. Strain selection and characterization of LL01-07 were conducted in vitro to evaluate their antimicrobial activity, carbohydrate fermentation ability, auto-aggregation, hydrophobicity, and acid resistance. An in vivo study in Sprague-Dawley rat model was conducted to evaluate its efficacy in oral wound healing.

Methods

Primary characterization and molecular identification

Gram staining and biochemical identification

The frozen stock of strain LL01-07 was previously isolated from dadih. Dadih was serially diluted in NaCl 0.9%, and the 1 × 10⁻² dilution was grown in blood agar under anaerobic conditions at 37 °C. Gram staining of the colony revealed Gram-positive short-chained cocci, then the colonies were purified and identified using VITEK 2 (bioMérieux, Marcy-l'Étoile, France), showing Lactococcus lactis subsp. lactis. The attribution of YPDH01-07 was based on the acronym of the dadih producer, for the different seven forms of colonies found in dadih. Oral pathogens used in this research were obtained from the American Type Culture Collection (ATCC). All Lactococcus strains were propagated in de Mann Rogosa Sharpe (MRS) (Merck) or blood agar medium (Oxoid). Oral pathogens were propagated in Brain Heart Infusion (BHI) medium (Oxoid), blood agar medium (Oxoid), and tripsic soy blood agar medium (Oxoid) and preserved as stock at −80 °C in tripsic soy broth-glycerol (TSB-glycerol).

Genotypic confirmation by PCR

These dadih-derived strains were initially re-identified by phenotypic attributes such as colony morphology and Gram staining. A PCR was performed to confirm the genotypic identification of the species from the bacterial culture stock. The primer set was based on Odamaki et al. and was intended to discriminate between L. lactis subsp. lactis and L. garviae (Table S1).²⁰

DNA was extracted from the bacterial culture using a boiling technique. The colony was dispersed in 1.5 ml of 1x PBS, centrifuged at 12000 rpm for 5 min, and the remaining pellet was resuspended in 500 μl 1x PBS, vortexed and centrifuged at 12000 rpm for 5 min. The pellet was resuspended in 200 μl 1× TE buffer and incubated for 10 min at 95 °C. Following incubation, the suspension was centrifuged at 12000 rpm for 2 min. The DNA supernatant was then obtained.

PCR was conducted to identify three forms of Lactococcus: L. lactis, L. garviae, and L. cremoris. The PCR reaction was preheated at 95 °C for 3 min, followed by 40 cycles of 95 °C for 10 s, annealing at 60 °C for 30 s, and extension at 72 °C for 30 s using a MA-6000 Real-Time Quantitative Thermal Cycler (Sansure, Beijing, China), and gel visualization in 20% agarose was performed using Geldoc (Biorad®, California, USA).

To confirm bacterial species, DNA sequencing was performed. PCR was conducted by using 16S rRNA general primers. DNA band was then purified and subjected to DNA sequencing, and the result was visualized and edited using SeqScanner 2. The initial quality assessment and necessary trimming were performed. The obtained sequences were aligned with the NCBI database using the BLAST algorithm.

Biosafety assessment

Hemolytic activity assay

The LL01-07 culture was streaked on blood agar plates containing 5% (v/v) sheep blood and incubated for 48 h at 37 °C. Staphylococcus pyogenes was used as a reference strain, a β-hemolysis strain, to evaluate hemolysis activity of cultures.²¹ The assessment of α or β-hemolysis was based on the development of a clear or greenish zone near the colonies.²²

Antibiotic resistance

The antibiotic sensitivity test was performed using the antibiotic disk diffusion method on agar blood based on the Clinical and Laboratory Standard Institute (CLSI) standards for Lactococcus lactis.²³ LL01-07 were cultured for 48 h at 37 °C, and the colony was adjusted to a solution of 0.5 McFarland (10⁸ viable cells) in 3 mL NaCl 0.9%. The diluted culture was streaked and evenly dispersed onto blood agar using sterile cotton swabs. Antibiotic discs were placed above the streaked culture and incubated for 48 h.²⁴ The antibiotics tested using the disc diffusion method were: gentamicin (10 μg), clindamycin (2 μg), ceftriaxone (30 μg), erythromycin (10 μg), oxoflaxin (1 μg), tetracycline (30 μg), and vancomycin (30 μg).

Functional characterization of probiotic properties

In probiotic strain selection, the functional ability that needs to be screened is the ability to resist environmental challenges, such as low gastric pH and competitive pathogen exclusion ability.²⁴

Carbohydrate fermentation assay

The carbohydrate fermentation activity of LL01-07 was evaluated based on its ability to hydrolyze mono- and disaccharides.²⁵ The reaction of carbohydrate fermentation is detected by basal medium color change when acid is produced after the addition of a single carbohydrate using bromothymol blue as a pH indicator.²⁶ The pH of the basal media ranged from 7.2 to 7.4. The accumulation of organic acids and, occasionally, carbon dioxide may occur during the growth of LAB in carbohydrate-containing media. For sugar fermentation, lactose, sucrose, maltose, and glucose were used. Glass tubes were filled with BHI broth and inoculated with L. lactis. After the addition of the pH indicator bromothymol blue and incubation at 35 ± 2 °C for 24 h, there was organic acid formation, then a decrease in pH causes a color change in the medium. Following the reduction of carbohydrate content by bacterial enzymes, the color changed to yellow. Formation of gas bubbles in the Durham's tube showed gas production.²⁵

Antimicrobial activity

As part of the bacterial competitive mechanisms, antimicrobial activity is evaluated. Antimicrobial activity is achieved by the production of bacteriocin or other antimicrobial substances such as hydrogen peroxide and organic acids.^24,27 The bacteriocin producer is immune to these peptides, but it can be active against other bacteria, including those from the same species.²⁴

The antimicrobial tests were done by agar well diffusion assay and agar disc diffusion assay. The cell-free supernatant (CFS) of each LAB was collected by centrifugation of the 5 ml overnight culture that had been aliquoted 1 ml each to five Eppendorf tubes at 12.000 rpm for 5 min then 600 μl of the upper supernatant was recollected into new tubes. The supernatant was filtered using a 0.20-μm cellulose acetate filter (Corning).^27,28 Each CFS was divided into two equal portions. One portion was unneutralized (pH 5), whereas the other portion was neutralized to pH 7 by adjusting the solution using 1 M NaOH. For the negative control, fresh uninoculated MRS broth (Merck) adjusted to pH 7 was used.²⁷

Each pathogenic bacterium was grown in BHI agar medium (Streptococcus mutans ATCC 25175 and Streptococcus oralis ATCC 6249), Blood agar medium (Staphylococcus aureus ATCC 25923 and Fusobacterium nucleatum ATCC 25586), and trypsic-soy blood agar medium (Porphyromonas gingivalis ATCC 33277) and adjusted to 0.5 McFarland (1.5 × 10⁸ CFU/ml). For agar well diffusion assay, about 100 μl oral pathogen was added to a soft BHI agar medium and mixed gently by moving the plate in a clockwise and counterclockwise motion. After the medium has solidified, 6-mm wells are made using a sterile cork borer. About 70 μl of the negative control, unneutralized CFS, and neutralized CFS were added into 6-mm diameter wells, and plates were incubated in a 4 °C refrigerator for 1 h to permit diffusion of the suspension into the agar.²⁷ Chlorhexidine gluconate 0.2% (Minosep^®) was used as a positive control in this experiment. Agar well diffusion assays were performed for S. mutans ATCC 25175, S. oralis ATCC 6249, and S. aureus ATCC 25923. The zone of inhibition was measured after incubation at 37 °C for 24 h.^27,29

Meanwhile, disc diffusion assays were conducted for F. nucleatum ATCC 25586 and P. gingivalis ATCC 33277, as they cannot grow in soft agar medium for agar well diffusion assays. Blank discs were saturated with 70 μl of negative control, unneutralized CFS, or neutralized CFS. The 0.5 McFarland pathogen suspension was spread evenly at the surface of the blood agar using a sterile cotton swab.²⁹ The disc was placed on the blood agar surface and incubated at 37 °C for 72 h for F. nucleatum ATCC 25586 and 5 days for P. gingivalis ATCC 33277. The incubation zone was calculated after the incubation period. The experiment was conducted in duplicate.

Auto-aggregation

The auto-aggregation assay was previously described by Mallappa et al. with some modifications.³⁰ Overnight cultures were centrifuged at 4,000 g for 20 min at room temperature and washed with PBS (Emsure^®, Merck). The pellets resuspended in PBS were adjusted to 10⁸ CFU/ml. The bacterial suspension was vortexed for 10 s, and using a spectrophotometer (Evolution 200 Thermo Scientific^®, Madison WI, USA), an absorbance at 600 nm was measured (A0). The suspension was then incubated at 37 °C for 3 and 22 h, and the absorbance of the upper fraction of the suspension was measured at 600 nm (At).²⁷ Each experiment was conducted in triplicate. The auto-aggregation percentage was enumerated as follows: Auto-aggregation (%) = (A₀ − A_t/A₀) × 100.

Hydrophobicity

Bacterial adhesion to the epithelium is important for colonization and infection.³¹ Hydrophobicity is a nonspecific and complex process of adhesion²⁸ and helps maintain the survival of bacteria in the gastrointestinal tract.²⁷ The hydrophobicity test in this study was based on the method by Vijayalakshmi et al.²⁷ Initially, the xylene solvent (J.T. Baker) was added to the bacterial suspension and thoroughly vortexed. After 3 h of incubation, the two-phase system was developed, the aqueous phase was taken out and the absorbance was measured at 600 nm. Hydrophobicity is an adhesion percentage based on the formula: [(A₀ − A)/A₀] × 100. A₀ is the initial absorbance of the bacterial suspension, and A is the absorbance after mixing with organic solvents. The result was calculated in three replicates, and it is expected that the optical density will decrease due to bacterial adhesion to the hydrocarbon layer.³¹

Acid resistance

Potential probiotics should be able to tolerate and survive acidic environments. The acid resistance of LAB supports its industrial use.²⁷ For oral probiotic use, the acid challenge was performed at pH 5 for 4 h because, in the oral cavity, the aciduric stage was observed in pH 4.5–5.5.³² The method used to investigate acid resistance was based on a study by Oh et al. with few modifications.³³ The isolates were cultured in MRS broth (Merck) at 37 °C for 24 h and harvested by centrifugation at 3000 rpm for 15 min. To prepare for culture suspensions, the pellets were resuspended in phosphate-buffered saline (PBS, pH 7.4). Each culture suspension was adjusted to pH 5 and incubated at 37 °C for 4 h. The strains were serially diluted using 0.85% (w/v) NaCl solution, and aliquots of samples were plated on blood agar at 0 and after 4 h. The culture was then incubated at 37 °C for 24 h, and the surviving cells were counted using the spread plate method to calculate the survival rate as follows:^13,18

Survival (%) = \frac{Number of surviving cells after incubation (CFU / ml) \times 100}{Initial number of cells before incubation (CFU / ml)}

In vivo study

Ethical approval was obtained from the Ethics Committee of the Faculty of Medicine, Universitas Indonesia, with ethical number No. KET 1368/UN2.F1/ETIK/PPM.00.02/2022. The experiment was conducted at the Animal Research Facilities, Indonesian Medical Education and Research Institute. Twelve seven-week-old male Sprague-Dawley rats, weighing about 250 grams, were randomly divided into two groups: normal control (N) (N = 6) and treatment group (N = 6) with high dose L. lactis subsp. lactis strain YPDH05 (LL05). The sample size was small because the number of animals used in the present study was those approved by the Ethics Committee, that applied the 3R principles (reduce, reuse, recycle). Rats were acclimatized for two weeks in 12-h light and dark cycles, 25 °C–27 °C temperature, 45%–65% humidity, lean bedding, and food and drink ad libitum. The LL05 in 1 × 10¹¹ CFU/ml dose (high dose) was given topically in the treatment group by rinsing with a 150 μl probiotic suspension using micropipettes every day since day one. Rats were held horizontally for 30 s to increase the contact time between LL05 and the oral mucosa. On the third day of the experiment, rats were anesthetized with 0,1 mL ketamine HCl 50 mg/ ml (Bernofarm) and 0,05 mL Xylazine 2% for injection (Xyla^®), and the buccal mucosa was scratched twice with a sterile 18-gauge needle to create a traumatic ulcer wound. The second upper molar and buccal commissure were used as an anatomical landmark to uniformly create the wound shape.

The lesion severity was evaluated clinically every two days and at the end of the experiment by Lima scoring: score 0: normal cheek pouch with absent or discrete erythema and hyperemia, no hemorrhagic areas, ulcerations, or abscess; score 1: moderate erythema and hyperemia, no hemorrhagic areas, ulceration, or abscess; score 2: severe erythema and hyperemia, presence of hemorrhagic areas, small ulceration, or scarred tissue, but no abscess; and score 3: severe erythema and hyperemia, presence of hemorrhagic areas, extensive ulcerations, and abscess of the buccal mucosa.³⁴ The experiment was conducted for 10 days.

Animal body weight was measured daily using a digital scale. The food intake was measured from day 3 to day 9 by weighing the remaining food and subtracting it from the known amount of food in each cage. On the 10^th day of experiments, all animals were euthanized using intraperitoneal 0,2 mL ketamine HCl 50 mg/ml (Bernofarm) and 0,1 mL Xylazine 2% for injection (Xyla^®). The death of animals was verified by the absence of heartbeat and respiration, and lack of reflexes. The study has followed the EQUATOR guidelines of ARRIVE for animal study.³⁵ The animals were given adequate care based on the guidelines of the ‘Guide for the Care and Use of Laboratory Animals, 8^th Edition.³⁶

Statistical analysis

In vitro experiments were carried out in duplicate or triplicate, and results were given as mean ± standard error of the mean (SEM). For in vivo experiment, unpaired t-test was conducted. GraphPad Prism version 10.0 (GraphPad Software, La Jolla, CA, United States) was used for statistical analysis. A p-value of less than 0.05 was considered significant.

Result

Primary characterization and molecular identification

The bacterial isolates used in this study originated from dadih in Agam, West Sumatra, Indonesia. The isolated dadih bacteria had L. lactis subsp. lactis as the most abundant strain, as identified with VITEK 2 (bioMérieux, Marcy-l'Étoile, France). There were seven isolates of LL01-07 based on the colony morphology that were stored as a TSB-glycerol stock at −80 ° C. Morphologically, LL01-07 were circular, raised, smooth, and shiny, 1–3 mm white colonies with an entire margin. Based on Gram staining, all strains are described as Gram-positive short-chain coccuses. The culture had a pleasant aroma similar to that of milk.

Initially, biochemical identification was performed using VITEK 2 (bioMérieux, Marcy-l'Étoile, France) to confirm the strain identification. VITEK 2 (bioMérieux, Marcy-l'Étoile, France) showed a low discrimination result between L. garviae and L. lactis subsp. lactis, and it was suggested to perform discrimination tests using the clindamycin susceptibility test. It showed that the strains were sensitive to clindamycin, indicating that they were closely related to L. lactis subsp. lactis.

Considering that the VITEK 2 (bioMérieux, Marcy-l'Étoile, France) result might be due to the similarity of biochemical properties, PCR was also performed to confirm the genotypic identification. The primer set was based on that of Odamaki et al. (Table S1).²⁰

The qPCR results showed that all isolates are positive for L. lactis subspecies but isolates of LL03 and LL06 were positive for L. garviae. All isolates were negative for L. lactis subsp. cremoris. Based on that, LL03 and 06 were omitted from the selection, and the remaining five isolates underwent further probiotic selection and characterization examination.

To confirm the genotypic identification, further examination by single-pass DNA sequencing was performed. The primers used for this analysis were 63F and 1387R. When visualized in SeqScanner 2, the initial quality assessment suggested that the samples originated from a single species. The sequence was investigated with the BLAST NCBI database, showing the Lactococcus lactis strain CAU 9592 16Sr RNA gene, partial sequence ranging from 85.09% in primer 63F to 86.31% in primer 1387R.

Biosafety assessment

Hemolytic activity assay

All isolates demonstrated no hemolytic activity compared to the reference strain Staphylococcus pyogenes, which showed a clear zone surrounding the colony or had β-hemolysis activity (Figure 1).

Figure 1.

Hemolytic activity assay of L. lactis subsp. lactis strain YPDH 01, 02 (A), 04, 05 (B), and 07 (C) with positive control (+) of Staphylococcus pyogenes.

Antibiotic resistance

The five isolates tested in this study were susceptible to gentamicin/CN, clindamycin/DA, ceftriaxone/CRO, erythromycin/E, ofloxacin/OFX, tetracycline/TE, and vancomycin/VA.

Functional characterization and probiotics properties of the isolates

Carbohydrate fermentation assay

Lactose, sucrose, maltose, and glucose were used for sugar fermentation. Glass tubes were filled with BHI broth (Oxoid) and inoculated with isolates. After the addition of pH indicator bromothymol blue and incubation at 35 ± 2 °C for 24 h, changes in color in tubes and the formation of gas bubbles in Durham's tube were observed. All isolates fermented all sugars. Initially, the medium pH was 7, but after incubation, it was adjusted to less than 5. Gas production in glucose tubes was found in LL04, 05, and 07.

Antimicrobial activity (Agar well diffusion and agar disc diffusion assay)

The pathogens used in agar well and disc diffusion assays were related to oral diseases such as S. mutans, S. oralis, F. nucleatum, P. gingivalis, and multisite infections (S. aureus). In addition, the antifungal activity of Candida albicans was also investigated. The zone of inhibition was calculated after incubation. There was no zone of inhibition observed for S. mutans, S. oralis, F. nucleatum, S. aureus, and C. albicans, either for unneutralized or neutralized L. lactis CFS. Meanwhile, there were slight zones of inhibition against P. gingivalis for L. lactis isolates as shown in Table S2. The highest activities were observed for LL04, 05, and 07; the next selection assay was performed for these isolates.

Auto-aggregation

For auto-aggregation properties, the selected isolates were observed for changes in optical density at 600 nm at 3 and 22 h. The highest rate of auto-aggregation was observed for LL05 at both 3 and 22 h (Table 1). Each experiment was conducted in triplicate and was expressed as means ± SEM (standard error of the mean).

Table 1.

Auto-aggregration abilities of probiotic strains.

Isolates	Time	% Autoagregration
LL04	3 h	14.07 ± 2.046
LL05	3 h	15.20 ± 1.744
LL07	3 h	14.17 ± 3.033
LL04	22 h	37.67 ± 7.140
LL05	22 h	54.17 ± 4.758
LL07	22 h	50.60 ± 4.050

Values are expressed as mean ± SEM (standard error of the mean).

Hydrophobicity

The cell surface hydrophobicity of all isolates toward xylene solvent was below 10%, with LL07 having the highest hydrophobicity rate (Table 2). The absorbance was measured after 3 h of incubation. Each experiment was conducted in triplicate. Data was expressed as means ± SEM.

Table 2.

Hydrophobicity (%) to xylene.

Isolates	Time	%Hydrophobicity
LL04	3 h	2.496 ± 1.642
LL05	3 h	1.128 ± 1.525
LL07	3 h	3.866 ± 1.24

Values are expressed as mean ± SEM (standard error of the mean).

Acid resistance test

Because the cell surface properties (auto-aggregation and hydrophobicity) of LL04 were below those of the other two isolates, the acid resistance test was only performed in LL05 and LL07. The results demonstrated that after an acid challenge for 4 h at pH 5, which simulated the oral cavity pH in the aciduric stage, the LL05 had a far better survival rate as shown in Table S3.

In vivo study

Clinical observation of wound healing capacity

All rats in each group survived the experiment without any signs of clinical toxicity, such as hair loss or diarrhea. Initially, all rats had the same oral lesion severity at score 2 (severe erythema and hyperemia, presence of hemorrhagic areas, small ulceration or scarred tissue, but no abscess), and the lesion gradually improved over time. The wound healing capacity was better in the control group since the improvement in oral severity scores was better on day 10 compared with the high-dose (1 × 10¹¹ CFU/ml) LL05 group. The oral lesion severity scores on days 4, 6, and 10 are illustrated in Figure 2 and summarized in Table S4.

Figure 2.

Lesion severity scores at days 4, day 6, and day 10 (right) in the control and treatment groups. Initial severity scores were 2 in both groups and gradually improved overtime.

Food intake and body weight changes

One day right after traumatic ulcer induction, there were decreases in food intake and body weight but those gradually increased again starting from day five as seen in Figure 3. This pattern is similar for both groups. All animals show increased body weight at the end of experiments.

Figure 3.

Gradual daily increase in body weight (A) and changes of food intake (B) of control (N = 6) and treatment (high dose LL05) groups (N = 6). Mean ± SD of bodyweight of control group (102.6 ± 0.613 (N = 6)) and treatment groups (102.9 ± 0.643 (N = 6)). Mean ± SD of food intake of control group (98.93 ± 0.899 (N = 6)) and treatment group (97.41 ± 0.995 (N = 6)) The mean body weight on day 1 and the mean food intake on day 3 (day 0 of investigation) is described as 100%. N, normal; N + LL HD, Normal + L. lactis subsp. lactis strain Y-PDH05 high dose (1 × 10¹¹ CFU/ml).

The treatment group showed a significantly higher body weight difference than the control group based on the t-test (p = 0.0199), as seen in Figure 3. On average, the percentage of body weight increase in the LL05 group is 2.9% and in the control group is 2.6%. At the end of the experiment, the treatment group had a 6.4% body weight increase, and the body weight increase of the control group was 6.2% compared to day one. Compared to the control group, the treatment group has a similar pattern of daily food intake, with a lesser amount of food intake seen in the LL05/treatment group (Figure 3). The treatment group also showed a greater reduction in average food intake (−3.25%) compared to the control group (−1.25%), but it was not statistically different based on the unpaired t-test.

Discussion

Phenotypic features of LL01-07 showed a circular, raised, smooth, and shiny, 1–3 mm white colony with an entire margin. Based on Gram staining, all strains were described as Gram-positive short-chain coccuses. The culture had a pleasant aroma, which is often perceived by probiotics that produce lactic acid related to the fermentation of dairy products. These morphological and aromatic features are similar to those of Lactococcus spp. described by other researchers.³⁷

For genotypic identification, RT-PCR and single-pass DNA sequencing were performed to confirm the genus, species, and strain levels of the isolates. Both results were positive for Lactococcus lactis subsp. lactis. The 16S-RNA sequencing shows 85–86% similarity to Lactococcus lactis strain CAU 9592. The RT-PCR showed that isolates of L. lactis subsp. lactis strains YPDH 03 and 06 were also positive for L. garviae; thus, these two strains were omitted for further selection and characterization.

Several assays were performed to determine the safety aspects, functional characteristics, and probiotic properties of dadih-derived isolates for oral cavity use. Some important characteristics of oral probiotics are oral pathogen growth inhibition, adherence ability, and carbohydrate fermentation (should be non-sugar fermenting to avoid pH decline in the oral cavity).¹⁴ Numbers of bacteria produce toxic by-products that could damage red blood cells.³⁸ There are three types of hemolysis: alpha hemolysis, beta hemolysis, and gamma hemolysis.^39,40 Alpha hemolysis is caused by the reduction of hemoglobin to methemoglobin, creating green or brown zones around the colonies.³⁸ Beta hemolysis indicates complete lysis of red blood cells, showing a clear zone surrounding the colonies.³⁹ Gamma hemolysis demonstrates a lack of hemolysis, showing no reaction in the medium.⁴⁰ Staphylococcus aureus ATCC 25923 as a positive control demonstrates beta hemolysis on blood agar.²⁶ A negative hemolytic activity is desired in probiotics because hemolytic bacteria might induce cell damage, leading to inflammation.³⁹ Alpha and beta hemolysis is considered as a virulence factor and can be found in many pathogens.¹ According to our study, LL01, LL02, LL04, LL05, and LL07 had no hemolytic potential (Figure 2).

One health risk concern regarding probiotics is the transmission of resistance determinants. In addition, probiotics contain high numbers of bacteria per serving. The oral cavity is ideal for bacterial growth because of its stable temperature, pH, and nutrient-rich environment. This condition promotes the transfer of the antibiotic resistance determinant.⁴¹ Antibiotic resistance is a vital concern in probiotics since it might relate to horizontal gene transfer from probiotics to host microbiota (either commensals or pathogens).⁴² It might be unsafe if the genes are transferred to oral pathogens such as S. mutans or P. gingivalis, further making the antibiotic treatment ineffective.⁴¹ In the current study, all isolates showed no antibiotic resistance towards gentamicin, clindamycin, ceftriaxone, erythromycin, ofloxacin, tetracycline, and vancomycin. Thus, the dadih-derived isolates were considered safe for the host. Lactococcus genus is classified as GRAS, and L. lactis cannot be considered as an opportunistic pathogen, as only two endocarditis cases were reported.⁴³

Lactococcus is lactic acid bacteria (LAB) that are facultative anaerobic, catalase-negative, and can transform sugar (importantly, glucose) into lactic acid.²⁴ The carbohydrate fermentation activity of LL01, LL02, LL04, LL05, and LL07 showed that all isolates can ferment glucose, sucrose, maltose, and lactose, causing a pH decline. Acid production due to carbohydrate fermentation induces dental caries. LL04, LL05, and LL07 exhibit heterofermentative traits, as demonstrated by gas production. The carbohydrate fermentation ability in this study was similar to L. lactis subsp. lactis, L. lactis subsp. cremoris, and other lactic acid bacteria as exhibited by the study of Gunkova et al. It showed that lactic acid bacteria are characterized by the ability to ferment maltose, lactose, sucrose, and glucose.²⁵

Based on our study results, the utilization of glucose, sucrose, maltose, and lactose by dadih-derived isolates indicates that they might compete with oral cariogenic bacteria in utilizing various carbohydrates. Even though Lactococcus is acidogenic and might be related to dental caries, it could be compensated by using dairy products to exert acidbuffering capacity.⁴⁴ Further study is needed to validate the cariogenic effect of LL01-07 and find the best approaches to compensate for it.

The antimicrobial activity of this strain was evaluated against oral bacteria (S. mutans, S. oralis, F. nucleatum, and P. gingivalis), also multi-site bacteria (S. aureus); and fungi (C. albicans). Pathogen exclusion can be achieved by nutrient competition, production of antimicrobial substances, and competition for epithelial adhesion. Antimicrobial substances produced by lactic acid bacteria include hydrogen peroxide, organic acids, low-molecular-weight antimicrobial substances, and bacteriocins.^24,27 This study used unneutralized CFS of dadih-derived bacteria to determine whether the antimicrobial activity was related to organic acid production, whereas neutralized CFS was used to detect whether the antimicrobial effects were not related to acid production (e.g antimicrobial peptide, and hydrogen peroxide).²¹ However, in our study, the neutralized and unneutralized CFS of isolates did not have antimicrobial activity against most of the oral bacteria tested and oral fungal C. albicans. The antimicrobial activity of the isolates appeared to be very minimal, with only very slight activity against P. gingivalis for LL01, 02, 04, 05, 07, and the effect was higher for neutralized CFS of LL04, LL05, and LL07, as seen in the inhibition zone diameter.

An antimicrobial activity of lactic acid bacteria is uncommon and difficult to screen. Mìnguez et al. revealed that only 52 of 169 LAB isolates from infant stool showed an inhibition zone larger than 10 mm against indicator strains.⁴⁵ Many LAB isolates were found ineffective against several species of pathogens.¹⁸

Detection of LAB antimicrobial activity could be affected by indicator strains and experiment. It is important to choose the right indicator strains because some bacteriocins have very limited targets. Several bacteriocins might also not be expressed under some circumstances. Production of bacteriocins needs optimization of the medium composition and the culture conditions. Diffusion, aggregation, concentration of bacteriocins, and cellular proteolytic activity can interfere with the measurement of bacteriocin activity in agar well diffusion assay.¹⁸

The cell surface properties of the isolates that promoted adherence were auto-aggregation and hydrophobicity. Auto-aggregation is the adhesion of bacteria to identical strains. This process is required for niche development, host colonization, and disease progression. Auto-aggregation, along with biofilm formation promotes antimicrobial resistance, survival, and persistence in certain hosts or niches. The protective effect of auto-aggregation is achieved by competitive exclusion and pathogen displacement.⁴⁶ One of the important characteristics of probiotics is their ability to adhere to epithelial cells and the mucosal surface, which is influenced by the interaction between the cell membrane and the interacting surface. Aggregation properties are related to this ability and hence enhance the survival of probiotics in the gastrointestinal tract.²⁷ The aggregation of bacteria in the liquid medium was observed as sediments at the bottom of the tube. The optical density alteration can be used to evaluate the auto-aggregation rate.⁴⁶ In our study, the auto-aggregation rate is small (below 20% at 3 h) but increased as the incubation time increased, indicating that auto-aggregation has a time-dependent manner. Similar to this study, Furtado et al. showed that, L. lactis DF04Mi has low auto-aggregation (12.2%).⁴⁷ Two new Lactococcus lactis subsp. lactis strains isolated from fermented food from India showed autoaggregation ranges from 15 to 25%.⁴⁸ Meanwhile, study by de Chiara et al. showed that L. lactis strains originating from natural whey starter culture showed 33.8% and 66.0% auto-aggregation values.¹⁸

Bacterial auto-aggregation is a complex and highly moderated interaction that occurs via specific adhesive interactions and can be modified transcriptionally, post-transcriptionally, and epigenetically. Auto-aggregation is vital to same-species bacterial colonization, which further impacts pathogen prevention and treatment.⁴⁶ Meanwhile, bacterial agglutination with other bacteria, known as coaggregation, may eliminate other bacteria. The low auto-aggregation rate in this study suggests that the adhesion ability of these isolates is low. The low levels of auto-aggregation and expected coaggregation with pathogens might play an important role in preventing the formation of biofilms, and in this way eliminating the pathogens from the gastrointestinal tract.⁴⁷ The coaggregation ability of these isolates to co-aggregate oral pathogens should be comprehensively examined in the future.

The method to observe cell surface hydrophobicity is the bacterial adhesion to hydrocarbons (BATH) test, later also known as the microbial adhesion to hydrocarbon (MATH) test.⁴⁹ A hydrophobicity experiment was conducted to qualitatively assess the bacterial surface polarity or non-polarity. It reflects the probiotic adhesion potential to the apolar surface of epithelia.²⁴ Xylene, an apolar solvent, exhibits cell surface hydrophobicity and hydrophilicity.³¹ In our study, the hydrophobicity was less than 10%, suggesting that the isolates have a low hydrophobicity according to the method of Ocana et al. that categorized hydrophobic characteristics into high (71 to 100%), medium (36 to 70%), and low (0 to 35%).⁵⁰ Other studies also showed low hydrophobicity values in L. lactis strains. Study by Tarazanova, et al. investigated the cell surface hydrophobicity of the bacterial cells to milk proteins from 55 L. lactis strains of which 25 were isolated from plants and 30 from a dairy environment. It revealed that 25 out of the 55 L. lactis strains have a low hydrophobicity, ranging from 0 to 20% for stationary and exponential growth phase.⁵¹

Lukic investigated the adhesion property of L. lactis subsp. lactis BGKP1 based on the presence of genes involved in aggregation (aggL) and possible interaction with mucin (mbpL).⁵² The hydrophobicity values were 3% (low hydrophobic) for construct-free plasmid strain of L. lactis subsp. lactis BGKP1, 15.7% (low hydrophobic) for strain with mbpL, and 48.3% (medium hydrophobic for strain with aggL. It demonstrated that aggL expression on the bacterial cell surface significantly increased hydrophobicity.⁵² This study suggests that specific genes are involved in bacterial hydrophobicity, so such studies are needed for our bacterial strains as well.

The hydrophobic potential is also organism- and strain-specific and influenced by the cell growth phase, surface chemistry of strains, and culture medium composition.¹⁸ The hydrophobic value of eight Lactococcus strains isolated from natural whey starter cultures varied from 25.5 to 78.8%. A good hydrophobicity has been shown for strains of different species, including Lactobacillus (43–79%), Pediococcus (51.3–79%), and Bifidobacterium (39–87%).¹⁸

Bacterial cell surfaces with glycol-(glycol-) proteinaceous material have a higher hydrophobicity, meanwhile, bacteria that contain polysaccharides have a hydrophilic surface.³¹ Bacteria with high hydrophobicity have fatty acids and other hydrophobic materials that help to adhere to fatty acids on the intestinal cell surface.³⁹ Hydrophobicity is a preliminary indication of mucus adhesion. Clinically, the process is more complex because it involves adherence proteins, cell surface carbohydrates distribution, and electrical charges.¹ In the oral cavity, cell surface hydrophobicity is related to adhesion, biofilm, coaggregation, and stereospecific interaction mechanisms.⁴⁹ Some studies reported correlations between cell hydrophobicity and intestinal epithelium adhesion, but others reported negative correlations. In vitro studies might not indicate the in vivo phenomenon and should be observed carefully.³³

After consumption, probiotic bacteria are exposed to oral and gastrointestinal acidity, further destroying probiotic bacterial cells and reducing their viability. Potential probiotic bacteria should be able to tolerate and survive in acidic environments. The acid resistance of LAB supports its industrial use.²⁷ In this study, only LL05 and LL07 had a better value of the cell surface properties; thus, the acid resistance assay was conducted in these two isolates. LL05 demonstrated significantly higher acid resistance than LL07 (75.2 and 2.4% respectively). L. lactis strains from natural whey exhibited high survival rate under gastrointestinal stress (95% to 100%) at pH 3.¹⁸ In another study, it was observed that the combination of a low, non-lethal acid stress (pH 5 or 5.5) and elevated temperature (37.5 °C) is lethal to L. lactis strain MG1363.⁵³

Lactococcus lactis growth is followed by lactic acid production, resulting in medium acidification and growth limitation at low pH. However, it is suggested that Lactococci have inducible responses to an acid pH. Mutans L. lactis strain MG1363 containing arl (acid-resistant locus), an acid-resistant insertional mutant of L. lactis, showed higher acid resistance.⁵³ Zhu et al. created overexpression of four genes in L. lactis NZ9000, concluding that the four ABC transporters overexpression can give acid stress tolerance on L. lactis.⁵⁴ Other LAB such as of Lactobacillus and Bifidobacterium genera could survive at extremely acidic pH (e.g. pH 2).¹⁸

In vitro, results are not sufficient for defining bacteria as probiotics.¹¹ Phenotypic characterization provides information about function in initial screening but is not a valid biomarker of probiotic functionality.¹³ Since results from in vitro studies may not always manifest in vivo situations,³³ in our research, in vivo experiment was conducted to provide more evidence of the functional probiotic properties.

A previous study that targeted the effect of Lactococcus lactis on periodontitis in rats showed that it might lower cytokine expression and reduce alveolar bone loss. It also demonstrated antimicrobial activity against P. gingivalis.¹⁹ Nevertheless, the wound healing score in our study did not improve in the LL05 group. In vivo experiments showed that supplementation with topical LL05 resulted in a statistically significant body weight increase compared with the normal group, even though the food intake was decreased. Although the result was statistically significant, the increase in body weight percentage was negligible (2.9%); thus, it does not necessarily imply that it is clinically meaningful. This suggests that topical probiotics can improve nutritional status or induce metabolic changes. The mechanism remains unclear, but it may be related to changes in the oral or gastrointestinal flora. Further validation is required to evaluate microbial and metabolic changes.

In this study, LL01, 02, 04, 05 and 07 were non-hemolytic, sensitive to antibiotics tested and have carbohydrate fermentation ability. The antimicrobial activity is better in LL04, LL05 and LL07, so that the other assays were performed only for these isolates. In autoagregration and hydrophobicity, all isolates have a low value, with higher value seen for LL05 and LL07. For acid resistance, LL05 has the best value. Based on the probiotic characterization result, LL05 was selected for an efficacy test for oral wound healing. No improvement in wound healing was seen in treatment groups, however, there was an increase in animal body weight. LL05 in this study did not show better wound healing effect. Each probiotic strain has a particular antimicrobial ability and disease specificity.¹⁸ The isolates need to be explored with other pathogens and also other oral diseases that might be more respond to them.

There were limitations in this study. The method of mucosal scratching has limitations, as a uniform wound is difficult to achieve, so in this experiment, we were trying to minimize it by standardizing the wound area. All rats survived and suffered no major morbidity.

The wound healing effect of Lactococcus strains was limited, suggesting that ingredients other than Lactococcus strains contained in dadih may have additive or synergistic effects. The whole dadih product has wound-healing or anti-inflammatory effects as shown in a previous colon inflammation study.¹⁶ The content of metabolic and dairy products may have therapeutic or preventive effects.¹⁶ The use of fresh dadih for future experiment may aid in elucidating the biochemical components that help reduce inflammation, however it best to perform after the standardized dadih production is achieved.

It is also important to consider that rather than the single bacterial strain, the multi-strain combination might enhance the health benefit.⁵⁵ For example, in vitro experiments of multi-strain probiotics containing Lactococcus lactis subsp. lactis CB460, L. lactis subsp. cremoris CB461, Streptococcus thermophilus, and Propionibacterium freudenreichii CB129 intensify anti-inflammatory cytokines in human peripheral blood mononuclear.⁵⁵ Strain combinations, such as dadih-derived L. lactis strains with Lactobacillus and Bifidobacterium, which have better hydrophobicity and acid resistance properties,¹⁸ might permit synergetic effects. Thus, exploration of a multi-strain probiotic is needed. In addition, in vivo experiments with more advanced designs, such as periodontitis models or oral mucositis induction, are needed to evaluate the potential of dadih-derived isolates for various oral health conditions. Finally, it could be interesting to test LL01-07 in combination with other adjunctive treatments such as ozone, photobiomodulation, and paraprobiotics.

Conclusion

The LL01, 02, 04, 05, and 07 isolates demonstrated an excellent safety assessment result based on hemolytic activity and antibiotic resistance properties. These isolates fermented lactose, glucose, maltose, and sucrose. Antibacterial properties of these dadih-derived bacteria were very limited towards oral pathogens. The auto-aggregation and hydrophobicity of LL04, LL05, and LL07 were low, while LL05 was superior in auto-aggregation and acid survival. The wound healing capacity of LL05 was not superior to that of the control group. The weight gain in the treatment group needs to be evaluated for a possibility of microbiome or metabolic change after strain administration. The predominant bacteria contained in dadih were previously thought to have a probiotic effect, but this study raised the possibility that this may not be the case. It must be considered that single strains might exhibit synergistic effects with other strains. The effects of other components of dadih besides the bacteria should also be investigated. Clinically, combination with adjunctive treatments can also be considered.

Supplemental Material

sj-docx-1-sci-10.1177_00368504251383108 - Supplemental material for Characterization of Lactococcus lactis subspecies lactis strain Y-PDH01-07 isolated from traditional Indonesian fermented milk (dadih) as an oral probiotic candidate

Supplemental material, sj-docx-1-sci-10.1177_00368504251383108 for Characterization of Lactococcus lactis subspecies lactis strain Y-PDH01-07 isolated from traditional Indonesian fermented milk (dadih) as an oral probiotic candidate by Ambar Kusuma Astuti, Andi Yasmon, Melva Louisa, Puspita Eka Wuyung, Jumpei Washio, Nobuhiro Takahashi and Yuniardini Septorini Wimardhani in Science Progress

Footnotes

Acknowledgments

The authors thank the Bacteriology Division, Molecular and Virology Division, and Micology Division of Microbiology Laboratory of Clinical Microbiology Department, and Animal Research Facilities, Faculty of Medicine, Universitas Indonesia for laboratory facilities and support.

ORCID iDs

Ambar Kusuma Astuti

Andi Yasmon

Melva Louisa

Puspita Eka Wuyung

Jumpei Washio

Nobuhiro Takahashi

Yuniardini Septorini Wimardhani

Ethical considerations

Ethical approval for this research was obtained from the Ethics Committee of the Faculty of Medicine, Universitas Indonesia approved at December 12, 2022, with ethical number No. KET 1368/UN2.F1/ETIK/PPM.00.02/2022. The animal experiment was performed at the Animal Research Facilities, Indonesian Medical Education and Research Institute.

Authors’ contribution

AKA, AY, ML, PEW, JW, NT, and YSW designed the protocol and interpreted the experimental results. AKA carried out experiments, conducted data analysis, and drafted the initial manuscript. All authors have reviewed and approved the final version of the manuscript.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This study was financially supported by a Publikasi Terindeks Internasional (PUTI) Q1 grant Universitas Indonesia [grant number NKB-431/UN2.RST/HKP.05.00/2023] awarded to Yuniardini S. Wimardhani in 2023. This work was part of a research study for a Doctoral Program at Universitas Indonesia.

Declaration of conflicting interests

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Data availability

The data supporting the findings of this study are available within the supplementary file.

Supplemental material

All supplemental material mentioned in the text is available in the online version of the journal.

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Supplementary Material

Please find the following supplemental material available below.

For Open Access articles published under a Creative Commons License, all supplemental material carries the same license as the article it is associated with.

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Characterization of Lactococcus lactis subspecies lactis strain Y-PDH01-07 isolated from traditional Indonesian fermented milk ( dadih ) as an oral probiotic candidate

Abstract

Objective

Methods

Results

Conclusions

Keywords

Introduction

Methods

Primary characterization and molecular identification

Gram staining and biochemical identification

Genotypic confirmation by PCR

Biosafety assessment

Hemolytic activity assay

Antibiotic resistance

Functional characterization of probiotic properties

Carbohydrate fermentation assay

Antimicrobial activity

Auto-aggregation

Hydrophobicity

Acid resistance

In vivo study

Statistical analysis

Result

Primary characterization and molecular identification

Biosafety assessment

Hemolytic activity assay

Antibiotic resistance

Functional characterization and probiotics properties of the isolates

Carbohydrate fermentation assay

Antimicrobial activity (Agar well diffusion and agar disc diffusion assay)

Auto-aggregation

Hydrophobicity

Acid resistance test

In vivo study

Clinical observation of wound healing capacity

Food intake and body weight changes

Discussion

Conclusion

Supplemental Material

sj-docx-1-sci-10.1177_00368504251383108 - Supplemental material for Characterization of Lactococcus lactis subspecies lactis strain Y-PDH01-07 isolated from traditional Indonesian fermented milk (dadih) as an oral probiotic candidate

Footnotes

Acknowledgments

ORCID iDs

Ethical considerations

Authors’ contribution

Funding

Declaration of conflicting interests

Data availability

Supplemental material

References

Supplementary Material